The Impact of Vitamin D Supplementation on the Expression of Klotho Protein in Diabetic Mice Retina

Klotho protein is a trans-membrane protein, with an antioxidant and anti-aging effect, in addition to its involvement in insulin resistance. Klotho was found to play a role in the normal retinal function in mice. Klotho -/- mice, have changes similar to those seen in age related macular degeneration. exogenous supplementation of vitamin D, increased the expression of Klotho protein in mice kidneys. In a meta-analysis, the results of 15 observational studies provided strong evidence that decreased serum 25(OH)D levels were associated with an increased risk of DR in type 2 diabetes patients. Four groups of DBA/2J mice, seven in each group, were included in our study. Group 1 was given citrate buffer only (the Streptozotozin solvent), group 2 had an induction of diabetes by infusion of Streptozotocin (STZ) as well as Propylene (Vitamin D solvent), group 3 had an induction of diabetes by STZ as well as vitamin D supplementation at the same time, and group 4 had diabetes induction by STZ and 3 weeks later given vitamin D. All the injections (of propylene glycol and of paricalcitrol) were repeated 3 times a week for 12 weeks or until mice were sacrificed. Seven mice were included in each group and all of them survived. 4 males and 3 females in each group except for group 3 that included 4 female and 3 male mice. All mice were sacrificed by ketamine, and 10 - 30 minutes later, their eyes were extracted including the optic nerve through disinsertion of lateral cantal ligament. The globe was later immersed in optimal cutting temperature (OCT) compound, and stored in ？70？C. From the second eye the retina was separated and fixated. slices were subjected to anti KLOTHO ab (ls-b7010, LSBio, France). Control slides were subjected to the fluorescent antibody only, without the anti Klotho abs. The slices were filmed by fluorescent microscope with a number of filters; Bright field, DAPI, MCHERRY and MRGE. The analysis of these films was done by imaging analysis application FIJI Image. The florescence scores of all groups were compared between each other using Kruskal-Wallis test followed by post-hoc test (Dunn’s test) The Mean fluorescence scores of groups 1, 2, 3 and 4 were 252995, 175420, 311890 and 311266, showing significant difference only between groups 2 and 4 (p = 0.038). In conclusion, late vitamin D supplementation significantly restored expression of Klotho protein in diabetic mice retina to its basal expression as in wild type mice.